A low-input Micro-C protocol for high-resolution 3D genome mapping

Standard Micro-C protocols typically require millions of cells, limiting their application to rare cell populations. Here, we present an optimized low-input Micro-C workflow that requires only 100 000 cells. By downsampling both our low-input dataset and a control dataset from 5 million cells to 120 million raw read pairs, we demonstrate that all key architectural features-Compartments, Topologically associating domains (TADs), and Chromatin loops-are reliably detected from as few as 100 000 cells. The low-input protocol achieved a high cis interaction ratio (96.1%) and low PCR duplication rate (3.0%), indicating high library complexity and low background noise. Applying this method to investigate acute CTCF (CCCTC-binding factor) degradation, we observed the loss of loops and TAD boundaries in CTCF-degraded samples, consistent with previous reports. Our optimized protocol enables nucleosome-resolution 3D genome mapping for sample-limited studies.